Hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke, and an emerging risk factor for Alzheimer's disease and vascular cognitive impairment. The basic mechanisms by which hyperhomocysteinemia predisposes to cerebral vascular disease are still poorly understood. We have developed dietary and genetic approaches to examine the mechanisms of vascular dysfunction in hyperhomocysteinemic mice. During the previous funding period of this program, we demonstrated that cerebral blood vessels are particularly susceptible to vascular dysfunction and hypertrophy during hyperhomocysteinemia and that the mechanism of these effects is dependent on vascular superoxide and loss of endothelium-derived nitric oxide. We now propose to use new strains of genetically-altered mice to define the molecular mechanisms which contribute to and protect the cerebral vasculature from oxidative stress, vascular dysfunction, and hypertrophy during hyperhomocysteinemia. Our specific goals are to determine the mechanistic role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS, in mediating cerebral vascular dysfunction and hypertrophy, and to test the hypotheses that superoxide dismutase (SOD) and peroxisome proliferator-activated receptor gamma (PPARy) protect from altered cerebral vascular structure and function during hyperhomocysteinemia. In Aim 1, we will use mice with altered expression of the ADMA hydrolyzing enzyme, dimethylarginine dimethylaminohydrolase (DDAH), to test the hypothesis that hyperhomocysteinemia produces adverse cerebral vascular effects through a mechanism involving ADMA. In Aim 2, we will use mice that overexpress, or are deficient in, SOD to test the hypothesis that SOD protects from cerebral vascular dysfunction and hypertrophy during hyperhomocysteinemia. In Aim 3, we will use mice with altered expression of wild-type or dominant-negative PPARy to test the 'hypothesis that PPARy protects from adverse cerebral vascular endpoints in the setting of hyperhomocysteinemia. A key feature of our experimental design is the use of mice with systemic or endothelial-specific expression of wild-type or dominant-negative gene constructs. This project has the potential to define the mechanisms of homocysteine's adverse effects on the cerebral circulation and to suggest novel therapeutic approaches to the prevention of cerebral vascular disease associated with hyperhomocysteinemia.